Elementary plant Biochemistry and Biotechnology (2+1)

Stage 1. Initiation of culture

1.Explant

• The nature of explant to be used for in vitro propagation is governed by the method of shoot multiplication. For enhanced axillary branching, only the explants which carry a pre-formed vegetative bud are suitable. When the objective is to produce virus-free plants from an infected individual it becomes necessary to start with sub-millimeter shoot tips. If the stock is virus-tested or virus eradication is not necessary, then the most suitable explant is nodal cuttings. Small shoot-tip explants have a low survival rate and show slow initial growth. Meristem tip culture may also result in the loss of certain horticultural characteristics which are controlled by the presence of virus, such as the clear-vein character of the Geranium cv. Crocodile. Generally, the clear vein character is transmitted in petiole-segment culture but not in shoot-tip culture.

2.Sterilization

• This operation needs to be done under sterilized conditions ie., inside Laminar airflow chamber. Special precautions need to be taken when explants are derived from field-grown materials, which is often necessary in cloning an elite tree. In such cases an ideal approach would be to take cuttings from the selected plant and grow them in greenhouse. It is necessary to treat the explants with suitable sterilizing agents such as 0.1% HgCl2 or sodium hypochlorite or other sterilants for 1- 10 minutes (the duration may vary with respect to the species, which needs to be standardized for each species). Then wash with double distilled sterilized water with five or six times Discarding the surface tissues from plant materials while preparing the explants also minimizes the loss of cultures due to microbial contamination. Appropriate size of the explants may be prepared under aseptic conditions and then it will be inoculated into the suitable medium.

3.Browning of medium

• A serious problem with the culture of some plant species is the exudation of phenolic compounds leached out from the cut surface of the explant. It turns the medium dark brown and is often toxic to the tissues. This problem is common with the adult tissues from woody species. This problem can be circumvented by adding activated charcoal, ascorbic acid, PVPP etc., into the medium.